João Miguel Freire

Receptors and routes of dengue virus entry into the host cells

Dengue is the most prevalent arthropod-borne viral disease, caused by dengue virus, a member of the Flaviviridae family. Its worldwide incidence is now a major health problem, with 2.5 billion people living in risk areas. In this review, we integrate the structural rearrangements of each viral protein and their functions in all the steps of virus entry into the host cells. We describe in detail the putative receptors and attachment factors in mammalian and mosquito cells, and the recognition of viral immunocomplexes via Fcγ receptor in immune cells. We also discuss that virus internalization might occur through distinct entry pathways, including clathrin-mediated or non-classical clathrin-independent endocytosis, depending on the host cell and virus serotype or strain. The implications of viral maturation in virus entry are also explored. Finally, we discuss the mechanisms of viral genome access to the cytoplasm. This includes the role of low pH-induced conformational changes in the envelope protein that mediate membrane fusion, and original insights raised by our recent work that supports the hypothesis that capsid protein would also be an active player in this process, acting on viral genome translocation into the cytoplasm.

Cell‐penetrating peptides: A tool for effective delivery in gene‐targeted therapies

The current landscapes of novel therapeutic approaches rely mostly on gene‐targeted technologies, enabling to fight rare genomic diseases, from infections to cancer and hereditary diseases. Although, reaching the action‐site for this novel treatments requires to deliver nucleic acids, or other macromolecules into cells, which may pose difficult tasks to pharmaceutical companies. To overcome this technological limitation, a wide variety of vectors have been developed in the past decades and have proven to be successful in delivering various therapeutics. Cell‐penetrating peptides (CPP) have been one of the technologies widely studied and have been increasingly used to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into cells. Despite the already proved huge potential that these peptide‐based approaches may suggest, few advances have been put to pharmacological or clinical use. This review will describe the origin, development, and usage of CPP to deliver therapeutic agents into cells, with special emphasis on their current application to gene‐therapies. Specifically, we will describe the current trials being conducted to treat cancer, gene disorders, and autoimmune diseases using CPP‐based therapies.

Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry

Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: Escherichia coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action were retrieved. Membrane interaction and permeabilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.

Apoptotic human neutrophil peptide-1 anti-tumor activity revealed by cellular biomechanics.

Cancer remains a major cause of morbidity and mortality worldwide. Although progress has been made regarding chemotherapeutic agents, new therapies that combine increased selectivity and efficacy with low resistance are still needed. In the search for new anticancer agents, therapies based on biologically active peptides, in particular, antimicrobial peptides (AMPs), have attracted attention for their decreased resistance development and low cytotoxicity. Many AMPs have proved to be tumoricidal agents against human cancer cells, but their mode of action is still controversial. The existence of common properties shared by the membranes of bacteria and tumor cells points to similar lipid-targeting mechanisms in both cases. On the other hand, anticancer peptides (ACPs) also induce apoptosis and inhibit angiogenesis. Human neutrophil peptide-1 (HNP-1) is an endogenous AMP that has been implicated in different cellular phenomena such as tumor proliferation. The presence of HNP-1 in the serum/plasma of oncologic patients turns this peptide into a potential tumor biomarker. The present work reveals the different effects of HNP-1 on the biophysical and nanomechanical properties of solid and hematological tumor cells. Studies on cellular morphology, cellular stiffness, and membrane ultrastructure and charge using atomic force microscopy (AFM) and zeta potential measurements show a preferential binding of HNP-1 to solid tumor cells from human prostate adenocarcinoma when compared to human leukemia cells. AFM also reveals induction of apoptosis with cellular membrane defects at very low peptide concentrations. Understanding ACPs mode(s) of action will certainly open innovative pathways for drug development in cancer treatment.

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.

Rethinking the capsid proteins of enveloped viruses: multifunctionality from genome packaging to genome transfection

Regardless of the debate on whether there is a place for viruses in the tree of life, it is consensual that they co‐evolve with their hosts under the pressure of genome minimization. The abundance of multifunctional viral structural proteins is a consequence of this pressure. The molecular key to multifunctionality is the existence of intrinsically disordered domains together with ordered domains in the same protein. Capsid proteins, the hallmark of viruses, are not exceptions because they have coexisting ordered and disordered domains that are crucial for multifunctionality. It is also frequent to find supercharged proteins (i.e. proteins for which the net charge per unit molecular mass is > +0.75/kDa) among viral capsid proteins. All flaviviruses having annotated proteins in the ExPASy Viralzone database have supercharged capsid proteins. Moreover, cell‐penetrating sequences/domains are frequent in viral proteins, even when they are not supercharged. Altogether, the findings strongly suggest that the ability to translocate membranes was acquired, conserved and optimized throughout the evolution of some viral proteins as part of their multifunctionality. The fitness of capsid proteins to translocate membranes carrying genomes was experimentally demonstrated with dengue virus capsid protein. This protein is potentially able to help the fusion process and translocate the RNA genome across the hemifused membrane formed by the viral envelope and the endosomal membrane. In addition, one of the cell‐penetrating domains of the capsid protein also has antibacterial activity. This may be reminiscent of parasitic bacteria–bacteria competition for the same host and shed light on the origins of enveloped viruses.

Shifting gear in antimicrobial and anticancer peptides biophysical studies: from vesicles to cells†

Despite the intensive study on the mechanism of action of membrane‐active molecules such as antimicrobial and anticancer peptides, most of the biophysical work has been performed using artificial model systems, mainly lipid vesicles. The use of these systems allows full control of the experimental parameters, and to obtain molecular‐level detail on the action of peptides, the correlation with biological action is intangible. Recently, several biophysical methodologies have been translated to studies using bacterial and cancer cells. Here, we review biophysical studies on the mechanism of action of antimicrobial and anticancer peptides performed directly on cells. The data in these studies allow to correlate vesicle‐based and cell‐based studies and fill the vesicle‐cell interdisciplinary gap. Copyright

Mining viral proteins for antimicrobial and cell-penetrating drug delivery peptides

MOTIVATION The need for more effective and safer pharmaceuticals is a persistent quest. Microbial adaptations create the need to permanently develop new antimicrobials (AMPs), for instance. Similarly, intracellular delivery of drugs is still a challenge and translocation of membranes for drug delivery is an area of intense research. Peptides can be used both as AMP drug leads and drug carrier systems for intracellular delivery. Multifunctional proteins are abundant in viruses but, surprisingly, have never been thoroughly screened for bioactive peptide sequences. RESULTS Using the AMPA and CellPPD online tools, we have evaluated the propensity of viral proteins to comprise AMP or cell-penetrating peptides (CPPs). Capsid proteins from both enveloped and non-enveloped viruses, and membrane and envelope proteins from enveloped viruses, in a total of 272 proteins from 133 viruses, were screened to detect the presence of potential AMP and CPP sequences. A pool of 2444 and 426 CPP and AMP sequences, respectively, were discovered. The capsids of flaviviruses are the best sources of these peptides reaching more than 80% of CPP sequence coverage per protein. Selected sequences were tested experimentally and validated the results. Overall, this study reveals that viruses form a natural multivalent biotechnological platform still underexplored in drug discovery and the heterogeneous abundance of CPP/AMP sequences among viral families opens new avenues in viral biology research.

Peptides as models for the structure and function of viral capsid proteins: Insights on dengue virus capsid

The structural organization of viral particles is among the most astonishing examples of molecular self-assembly in nature, involving proteins, nucleic acids, and, sometimes, lipids. Proper assembly is essential to produce well structured infectious virions. A great variety of structural arrangements can be found in viral particles. Nucleocapsids, for instance, may display highly ordered geometric shapes or consist in macroscopically amorphous packs of the viral genome. Alphavirus and flavivirus are viral genera that exemplify these extreme cases, the former comprising viral particles structured with a T = 4 icosahedral symmetry, whereas flavivirus capsids have no regular geometry. Dengue virus is a member of flavivirus genus and is used in this article to illustrate how viral protein-derived peptides can be used advantageously over full-length proteins to unravel the foundations of viral supramolecular assemblies. Membrane- and viral RNA-binding data of capsid protein-derived dengue virus peptides are used to explain the amorphous organization of the viral capsid. Our results combine bioinformatic and spectroscopic approaches using two- or three-component peptide and/or nucleic acid and/or lipid systems.

siRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model

&NA; Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr‐Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA‐based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell‐penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer‐related signaling processes. Both pepM and pepR penetrate the positive Bcr‐Abl+ Cell Line (BV173). Five in silico designed anti‐Bcr‐Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr‐Abl downregulation kinetics (48 h to 168 h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome‐wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr‐Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120 h. Both pepM and pepR showed downregulation effects on proliferative CML‐related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM‐siRNA conjugates lowered Bcr‐Abl gene expression levels more extensively than conventional siRNA delivery technologies and perturbed leukemogenic cell homeostasis, hence revealing their potential as novel alternative scaffolds for CML therapy. Graphical abstract A siRNA‐based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell‐penetrating peptides (CPPs) derived from the dengue virus capsid (DENV C) protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer‐related signaling processes. Maximal Bcr‐Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120 h. Both pepM and pepR showed downregulation effects on proliferative CML‐related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. Figure. No caption available.